Q-Band Pulsed EPR Spectrometer

我们小组研究了几个重要的膜蛋白复合体的结构、功能和动力学。电子自旋共振波谱技术，特别是脉冲电子-电子双共振技术是我们研究的主要生物物理工具。由于几个原因，对膜蛋白进行这样的实验是非常具有挑战性的。低表达水平往往导致样本量较少。此外，当结合到膜蛋白上时，自旋标记的相记忆时间显著减少。这些限制再加上传统矩形微波脉冲激励带宽较窄，导致灵敏度较低，也使更长距离(&gt；5 nm)的测量变得极其困难。对于膜蛋白结构生物学，越来越多的证据表明天然脂环境对蛋白质折叠、结构和功能起着至关重要的作用。我们工作组的主要活动之一是发展和应用脉冲电子-电子双共振原位研究膜蛋白。由于其他一些原因，如背景标记、自旋标记的不稳定性和低蛋白质表达等，这甚至更具挑战性。在过去的几年里，高性能脉冲光谱仪的问世大大提高了膜蛋白实验的灵敏度。该装置配备了高功率微波放大器和任意波形发生器，为膜蛋白的新研究提供了前所未有的机会。利用这样的装置，我们的目标是表征构成不同膜转运蛋白复合体功能基础的构象异质性和平衡动力学。将测试新的方法，包括不同的自旋标记、标记策略和原位电子自旋共振光谱的样品制备方案。稍后，这些方法将被应用于研究蛋白质折叠β-桶组装机械复合体和革兰氏阴性细菌的脂多糖运输系统。这两个系统在革兰氏阴性细菌中都是必不可少的和保守的，因此是新药非常受欢迎的靶点。此外，我们还一直在研究初级和次级活性膜转运蛋白的底物转运机制。利用ATP结合盒导出器TmrAB，我们证明了在三个单独的结构域上独立观察构象平衡的可行性。利用质子偶联的富马酸同向转运体SLC26Dg，我们用脉冲电子-电子双共振约束法确定了蛋白质脂质体中的二聚体结构。我们的目标是进一步阐明蛋白质-底物和蛋白质-脂质相互作用的细节，以及探索这些转运蛋白在底物转运过程中热力学参数的变化。

Our group investigates the structure, function, and dynamics of a few important membrane protein complexes. Electron spin resonance spectroscopy techniques, especially pulsed electron-electron double resonance is used as the major biophysical tool in our research. Such experiments on membrane proteins are very challenging for several reasons. The low expression levels often lead to small sample quantities. In addition, when attached to membrane proteins, the phase memory time of the spin labels get significantly reduced. These limitations combined with the narrow excitation bandwidth of conventional rectangular microwave pulses lead to poor sensitivity and also make the measurement of longer distances (> 5 nm) extremely difficult. For membrane protein structural biology, there is increasing evidence for the vital role of native lipid environment for protein folding, structure, and function. One of the major activities of our working group is to develop and apply pulsed electron-electron double resonance for studying membrane proteins in situ. For a few additional reasons such as the background labeling, instability of the spin labels, and the low protein expression, etc., this is even more challenging. Over the past few years, the availability of a high-performance pulsed spectrometer has greatly enhanced the sensitivity for such experiments with membrane proteins. This device is equipped with a high-power microwave amplifier and an arbitrary waveform generator, which together offer unprecedented opportunities for novel investigations in membrane proteins. Using such a device, we aim to characterize the conformational heterogeneity and the equilibrium dynamics that form the basis of function in different membrane transport protein complexes. New approaches, including different spin labels, labeling strategies, and sample preparation protocols for in situ electron spin resonance spectroscopy will be tested. Later those approached will be applied to study the protein folding beta-barrel assembly machinery complex and the lipopolysaccharide transport systems of Gram-negative bacteria. Both of these systems are essential and conserved in Gram-negative bacteria and therefore are highly sought-after targets for new drugs. In addition, we have been studying the substrate translocation mechanism for primary and secondary active membrane transporters. With the ATP Binding Cassette exporter TmrAB, we showed the feasibility to independently observe the conformational equilibria at three individual domains. With the proton-coupled fumarate symporter SLC26Dg, we determined the dimer structure in proteoliposomes using pulsed electron-electron double resonance constraints. We aim to further elucidate the details of protein-substrate and protein-lipid interactions as well to explore the changes in the thermodynamic parameters during substrate translocation in these transporters.